Our overall objective is to develop approaches for effective vaccination against pulmonary tularemia. Our strategy will involve an innovative combination of vaccine approaches that will exploit TLR/NLR agonists identified in Subproject 2 and anti-oxidant F tularensis (Ft) mutants developed in Subproject 3. During the previous funding period, we made considerable progress (see Program Overview), including the demonstration that significant protection in C57BL/6 mice against the highly virulent type A strain of Ft, SchuS4, can be induced by i.n. inoculation of FcR-targeted inactivated LVS (\Ft). This is the first case to our knowledge in which an inactivated vaccine has provided protection against Ft SchuS4. In addition, anti-oxidant mutants (AAO) of Ft were found to induce partial protection against SchuS4 in the highly sensitive C57BI/6 mouse strain. In this renewal application, we will further optimize conditions for mucosal vaccination, characterize the effects of pulmonary vaccination, and define the cellular and humoral mechanisms responsible for protective immunity against SchuS4. Specifically, we will: 1) Determine the ability of i.n. vaccination with FcR-targeted immunogens in combination with use of anti-oxidant mutants (AAO) of Ft generated in Subproject 3, and /or TLR/NLR agonists identified in Subproject 2, to maximally enhance the immune response to, and levels of protection against, mucosal (i.n.) challenge with Ft SchuS4 at challenge doses up to 20,000 CPU; 2) Establish the impact of Ft LVS, AAO Ft LVS and AAO Ft SchuS4, both in live and inactivated forms, on processing and presentation by M0 and DC of non-targeted and FcRtargeted Ft antigen in the presence or absence TLR/NLR agonists; and 3) Define the effector mechanisms responsible for enhanced protection after i.n. vaccination with protective immunogens identified in Aim 1 including testing the postulate that synergy between humoral and cellular immune mechanisms is required for induction of effective protection. The roles of TLR/NLRs and ROS/RNS in both inductive and effector phases will also be examined by using genetically deficient mice and specific agonists/antagonists, in consultation with the PLs of subprojects 2 and 3. The results of subproject 1 will allow the design of new mucosal vaccination platforms for effective biodefense against infection with virulent Ft and will provide novel insight into the pulmonary immune mechanisms that are responsible for protection against respiratory tularemia.